False-negative Reactions Research Articles

Baseline antibody titres against Salmonella typhi in apparently asymptomatic HIV positive individuals in a tertiary care hospital

Enteric fever is common in tropical regions and is caused by Salmonella enterica serotype typhi (S typhi ). For diagnosis of enteric fever, the Widal test is the most widely used test after blood culture. In HIV infected individuals false positive and false negative Widal reactions are common. The result is variable titres and baseline titres that are unusual in this patient population. This study was done to determine the baseline antibody titres for S typhi among HIV infected individuals. Average baseline antibody titres against O and H antigens of S typhi were measured by standard Widal test in 200 HIV positive asymptomatic individuals, as well as 200 age and sex-matched controls. The results were compiled and statistically analysed. A total of 84 (42%) of the cases had an H antibody titre of >1:20 and 105 (52.5%) had a titre of >1:20 against O antigen. This implies that positive titre of H and O antigen is significantly associated with HIV positive cases with P<0.001. Correlation of CD4 count with antibody titres shows that there is no significant association between CD4 counts and antibody titres against either H (P=0.634) or O antigen (P=0.765). This study shows that HIV infected individuals had increased titres of antibodies against S typhi from the baseline. This indicates a need for evaluation of current cut-off values of diagnostic titres for this group. We also suggest that it is best to perform baseline titres against S t yphi for each patient at the time of diagnosis of HIV status, and to use this for future reference.

Comparing the performance of traditional non-treponemal tests on syphilis and non-syphilis serum samples

The goal of the present study was to determine the performance of two traditional non-treponemal tests for syphilis. Syphilis sera (n = 209) included different stages of disease, and control sera (n = 247) were from patients with tumours, leprosy, systemic lupus erythematosus, hepatitis, pregnant women and healthy individuals. Treponema pallidum ELISA, Treponema pallidum particle agglutination and rapid treponema-specific tests were used as gold standards. Rapid plasma reagin or toluidine red unheated serum test had a sensitivity and specificity of over 95%. False-negative reactions of rapid plasma reagin and toluidine red unheated serum test were observed mainly in primary and latent syphilis cases, and false-positive reactions were present in systemic lupus erythematosus, hepatitis-infected patients. Overall, both non-treponemal tests had high sensitivities and specificities making the assays attractive as screening tests for syphilis. When examined on WHO reference serum samples and based on lower limits of detection, non-treponemal tests were less sensitive than treponema-specific tests.

The evolutionary divergence of Shiga toxin-producing Escherichia coli is reflected in clustered regularly interspaced short palindromic repeat (CRISPR) spacer composition.

The Shiga toxin-producing Escherichia coli (STEC) strains, including those of O157:H7 and the "big six" serogroups (i.e., serogroups O26, O45, O103, O111, O121, and O145), are a group of pathogens designated food adulterants in the United States. The relatively conserved nature of clustered regularly interspaced short palindromic repeats (CRISPRs) in phylogenetically related E. coli strains makes them potential subtyping markers for STEC detection, and a quantitative PCR (qPCR)-based assay was previously developed for O26:H11, O45:H2, O103:H2, O111:H8, O121:H19, O145:H28, and O157:H7 isolates. To better evaluate the sensitivity and specificity of this qPCR method, the CRISPR loci of 252 O157 and big-six STEC isolates were sequenced and analyzed along with 563 CRISPR1 and 624 CRISPR2 sequences available in GenBank. General conservation of spacer content and order was observed within each O157 and big-six serogroup, validating the qPCR method. Meanwhile, it was found that spacer deletion, the presence of an insertion sequence, and distinct alleles within a serogroup are sources of false-negative reactions. Conservation of CRISPR arrays among isolates expressing the same flagellar antigen, specifically, H7, H2, and H11, suggested that these isolates share an ancestor and provided an explanation for the false positives previously observed in the qPCR results. An analysis of spacer distribution across E. coli strains provided limited evidence for temporal spacer acquisition. Conversely, comparison of CRISPR sequences between strains along the stepwise evolution of O157:H7 from its O55:H7 ancestor revealed that, over this ∼7,000-year span, spacer deletion was the primary force generating CRISPR diversity.

An antiretroviral drug-naïve human immunodeficiency virus-1 infected woman with a persistent non-reactive proviral deoxyribonucleic acid polymerase chain reaction: a case report.

IntroductionReplication of the human immunodeficiency virus involves an obligatory step of reverse transcription of the viral ribonucleic acid genome into a double-stranded deoxyribonucleic acid, and subsequent integration of the deoxyribonucleic acid into the human chromatin to form the proviral deoxyribonucleic acid. This proviral human immunodeficiency virus deoxyribonucleic acid is a critical marker for the diagnosis of acute infections, mother-to-child transmissions and for the confirmation of indeterminate serological reactions. We describe a case of a human immunodeficiency virus positive woman, naïve to antiretroviral treatment, who was persistently negative for human immunodeficiency virus proviral deoxyribonucleic acid polymerase chain reaction. This observation, to the best of our knowledge, is the first time that it has been described in Africa.Case presentationA 28-year-old Gabonese woman living in Cameroon requested a human immunodeficiency virus diagnosis in our laboratory. She had an unprotected heterosexual contact 6 months earlier while on vacation in Gabon. The request for a human immunodeficiency virus test was as a result of apprehensions developed after the exposure episode. Human immunodeficiency virus serological examinations were ambiguous and confirmatory tests (including human immunodeficiency virus proviral deoxyribonucleic acid polymerase chain reaction) were carried out. Apart from the human immunodeficiency virus proviral deoxyribonucleic acid polymerase chain reaction that was persistently negative, all other polymerase chain reactions carried out were positive. The deoxyribonucleic acid sequences have been submitted to the GenBank database with accession numbers: KC626022, KC626023 and KC626024 for the protease, reverse transcriptase and gp41 genes respectively.ConclusionThe persistently negative human immunodeficiency virus proviral deoxyribonucleic acid polymerase chain reaction in a person with a confirmed human immunodeficiency virus infection is of immense importance in the human immunodeficiency virus diagnostic field. This could highlight the fact that cases of false negative human immunodeficiency virus proviral deoxyribonucleic acid polymerase chain reactions exist especially with the high genetic variations observed with human immunodeficiency virus. The challenges presented by such false negative tests in the identification of acute infections, mother-to-child transmissions and the confirmation of indeterminate serological reactions are daunting. These data therefore would be invaluable especially to clinicians in Africa where non-B human immunodeficiency virus subtypes circulate.

FENÓMENO PROZONA EM SÍFILIS SECUNDÁRIA. A IMPORTÂNCIA DA COMUNICAÇÃO ENTRE O CLÍNICO E O LABORATÓRIO

O fenómeno prozona consiste na obtenção de um resultado falso negativo num teste de diagnóstico imu- nológico devido a elevada concentração do título de anticorpos produzidos pelo doente. Esta concentração excessiva pode levar à não formação de complexos antigénio-anticorpo e pode ocorrer em doenças como a sífilis ou a brucelose. Os autores descrevem o caso de um doente do sexo masculino observado no Serviço de Urgência por erupção sugestiva de sífilis secundária ou linfoma cutâneo. Dado que o primeiro RPR foi negativo, o doente foi internado para confirmar ou excluir a segunda hipótese de diagnóstico. As biópsias cutâneas realizadas não revelaram linfoma, mas foram sugestivas de sífilis. O laboratório foi contactado para repetir a titulação sob protocolo dilucional o que permitiu a obtenção de VDRL positivo (título 1/256). Este caso ilustra a importância do contacto próximo entre o clínico e o laboratório para evitar resultados de sífilis falsa- mente negativos, tais como os que são gerados pelo fenómeno prozona.PALAVRAS-CHAVE – Sífilis; Fenómeno Prozona.

Guidelines for the investigation and management of mantle cell lymphoma.

The guideline group was selected to be representative of UK-based medical experts and patients representatives. Ovid MEDLINE, EMBASE and NCBI Pubmed were searched systematically for publications in English from 1980 to 2011 using the MeSH subheading 'lymphoma, mantle cell' and 'lymphoma, mantle cell' as a keyword, as well as all subheadings. In addition, all references to mantle cell lymphoma in the World Health Organization Classification of Tumours of Haematopoietic and Lymphoid Tissue (Swerdlow et al, 2008) and the British Committee for Standards in Haematology (BCSH) Guideline: Best Practice in Lymphoma Diagnosis and Reporting (Parker et al, 2010) have been included. The writing group produced the draft guideline, which was subsequently revised by consensus by members of the Haemato-oncology Task Force of the BCSH. The guideline was then reviewed by a sounding board of approximately 50 UK haematologists, the BCSH and the British Society for Haematology Committee and comments incorporated where appropriate. The 'GRADE' system was used to quote levels and grades of evidence, details of which can be found in Appendix I. The objective of this guideline is to provide healthcare professionals with clear guidance on the investigation and management of patients with mantle cell lymphoma. The guidance may not be appropriate to patients with other lymphoma sub-types and in all cases individual patient circumstances may dictate an alternative approach.

Decapitation Improves Detection of &lt;I&gt;Wolbachia pipientis&lt;/I&gt; (Rickettsiales: Anaplasmataceae) in &lt;I&gt;Culex pipiens&lt;/I&gt; (Diptera: Culicidae) Mosquitoes by the Polymerase Chain Reaction

Polymerase chain reaction (PCR) is often used to detect microorganisms, pathogens, or both, including the reproductive parasite Wolbachia pipientis (Rickettsiales: Anaplasmataceae), in mosquitoes. Natural populations of Culex pipiens L. (Diptera: Culicidae) mosquitoes are infected with one or more strains of W. pipientis, and crosses between mosquitoes harboring different Wolbachia strains provide one of the best-known examples of cytoplasmic incompatibililty (CI). When we used PCR to monitor Wolbachia in the Buckeye strain of Culex pipiens, and in a Wolbachia-cured sister colony obtained by tetracycline treatment, we noted false negative PCR reactions with DNA samples from infected mosquitoes; these results were inconsistent with direct microscopic observation of Wolbachia-like particles in gonads dissected from mosquitoes in the same population. Assays with diluted template often improved detection of positive samples, suggesting that DNA prepared from whole mosquitoes contained an inhibitor of the PCR reaction. We reconciled discrepancies between PCR and microscopy by systematic measurement of the PCR reaction in the presence of an internal standard. Mosquito decapitation before DNA extraction restored the reliability of the PCR reaction, allowing accurate determination of Wolbachia infection status in infected and tetracycline-cured mosquito populations, consistent with microscopic examination. Using PCR primers based on the Tr1 gene, we confirmed that the Wolbachia infection in the Buckeye strain of Culex pipiens belongs to the genotype designated wPip1. Finally, to explore more widely the distribution of PCR inhibitors, we demonstrated that DNA isolated from the cricket, Acheta domesticus (L.); the beetle, Tenebrio molitor L.; the honey bee, Apis mellifera L.; and the mosquito, Anopheles punctipennis Say also contained PCR inhibitors. These results underscore the importance of measuring the presence of inhibitors in PCR templates by using a known positive standard, and provide an approach that will facilitate use of PCR to monitor environmental samples of mosquitoes that harbor endosymbionts or pathogenic organisms.

Association of platelet count and serological markers of dengue infection-importance of NS1 antigen

Introduction: Dengue is an acute viral infection with potential fatal complications. Specific antibody detection has been the mainstay of diagnosis which is prone for both false positive and false negative reactions. The newer parameter NS1 appears to be highly specific and reliable for diagnosis of dengue infection from the first day of fever. Platelet count is the only accessory test for diagnosis of dengue infection in the peripheral laboratories. Therefore, we tried to evaluate the association of platelet counts against NS1 and IgM/IgG in dengue infections. Materials andMethods: Serum samples from clinically suspected dengue cases were tested for NS1, IgM and IgG by immunochromatography-based test. Platelet counts were obtained for all positive cases and 150 dengue seronegative cases of fever that served as controls. Test results of dengue-specific parameters were compared against platelet counts. The proportions obtained were compared by Standard error of the difference between the proportions (SEP test). Results: Of 2104 samples tested, 320 were positive for one or more dengue parameters. Of the 320, 95 were positive for NS1 only, 161 showed IgM only while 9 showed IgG only. More than one marker was detected in the remaining 55 samples. Thrombocytopenia was more consistently associated whenever NS1 was detected compared to antibody detection (P value <0.001). Conclusions: Inclusion of NS1 in the diagnosis of dengue increases the detection rate significantly. In cases of fever, thrombocytopenia is more consistently found in dengue positive rather than dengue negative subjects. It correlates well when NS1 and IgM are detected simultaneously.

178-P The geometry of HLA typing reactivity patterns: Doing away with false-positive and false-negative reactions

178-P The geometry of HLA typing reactivity patterns: Doing away with false-positive and false-negative reactions

ALERGIA CUTÂNEA EM CRIANÇAS

No diagnóstico de dermatite de contacto alérgica é fundamental, tal como nos adultos, a história e loca- lização das lesões, mas alguns produtos e/ou hábitos característicos das crianças e adolescentes podem ser respon- sáveis por quadros clínicos pouco usuais. Os alergenos mais importantes na criança são os metais, como o crómio (calçado) e o níquel (por vezes associado ao cobalto ou paládio; este último também responsável por reacções gra- nulomatosas de contacto), sobretudo nas jovens, dada a popularidade dos adornos baratos. Recentemente, surgiram entre os jovens outras fontes de sensibilização, como os telemóveis. Outros potenciais alergenos são os ingredientes de produtos tópicos, sobretudo antissépticos; o mercúrio e derivados ainda são utilizados nalguns países, mas as re- acções alérgicas, mesmo em crianças pequenas, não são em geral clinicamente relevantes. Cosméticos, em particular de limpeza cutânea, podem originar sensibilização a componentes das fragrâncias ou conservantes. Derivados da borracha, muitas vezes responsáveis por dermatite ao calçado ou às fraldas, resinas e plantas também podem ser fonte de sensibilização. Adicionalmente, alguns alergenos ocupacionais (p. ex, cabeleireiras, construção civil e me- talúrgicos) também ocorrem em adolescentes. A alteração da legislação, referente à presença de alergenos em pro- dutos comuns, pode levar à redução da incidência de alergia de contacto em crianças (p. ex, ao níquel nos adornos e telemóveis e às tintas capilares em menores de 16 anos). O uso de p-fenilenodiamina nas tatuagens temporárias de henna continua a ser um problema. As provas epicutâneas em crianças são seguras; a maioria dos autores con- sideram as reacções irritativas pouco frequentes (excepto em atópicos, em particular a metais) e podem utilizar-se as mesmas concentrações que no adulto. No entanto, não se pode excluir a possibilidade de reacções falso+ ou falso- e, na suspeita de potencial irritativo, devem também testar-se concentrações inferiores. Dada a reduzida área cutânea para testes, a menor exposição ambiental a alguns alergenos e a hipermobilidade das crianças pequenas, alguns autores recomendam utilizar séries standard reduzidas, embora outros alertem para o risco de se ignorarem muitas reacções. De qualquer modo, é importante ter em conta a história e o quadro clínico e testar, sempre que necessário, outros possíveis alergenos e produtos pessoais.PALAVRAS-CHAVE – Dermatite; Alergia de Contacto; Criança; Alergenos; Hipersensibilidade.

Point-of-care blood test for ketones in patients with diabetes: primary care diagnostic technology update

#### Clinical Question In patients with diabetes, does a point-of-care test for blood ketones improve the diagnosis and management of diabetic ketoacidosis compared to standard practice? Earlier diagnosis of capillary blood ketones associated with hyperglycaemia facilitates the prevention of ketoacidosis, as well as prompt treatment.1 Urine ketone strips are semi-quantitative and measurements do not accurately reflect current conditions if the urine has been in the bladder for several hours (for example, overnight) before testing. Bottled strips can lose their accuracy if the bottle has been open for several months. Results can also be affected by medications, giving false-positive results in the presence of drugs containing sulphydryl groups, or false-negative reactions in the presence of ascorbic acid. In some cases, patients may also be unable to provide a urine sample (for example, due to confusion). Three portable point-of-care (POC) blood ketone meters were identified, which require ∼1 μl of blood and a test time of ∼10 seconds: the Precision Xtra® (called Optium Xceed in the UK; Abbott Diabetes Care; detection range 0.1-6 mmol/l); the Nova Max® PLUS blood glucose and beta-ketone monitoring system (Nova Biomedical; detection range 0.1-8 mmol/l), and the GlucoMen® Lx Plus (A. Menarini Diagnostics; detection range 0.1-8 mmol/l). DKA remains a leading cause of hospitalisation and the main cause of morbidity and death in children and adolescents with …

Exogenous Controls Increase Negative Call Veracity in Multiplexed, Quantitative PCR Assays for Phakopsora pachyrhizi.

Quantitative polymerase chain reaction (Q-PCR) utilizing specific primer sequences and a fluorogenic, 5'-exonuclease linear hydrolysis probe is well established as a detection and identification method for Phakopsora pachyrhizi and P. meibomiae, two rust pathogens of soybean. Because of the extreme sensitivity of Q-PCR, the DNA of single urediniospores of these fungi can be detected from total DNA extracts of environmental samples. However, some DNA preparations unpredictably contain PCR inhibitors that increase the frequency of false negatives indistinguishable from true negatives. Three synthetic DNA molecules of arbitrary sequence were constructed as multiplexed internal controls (ICs) to cull false-negative results by producing a positive signal to validate the PCR process within each individual reaction. The first two, PpaIC and PmeIC, are a single-stranded oligonucleotide flanked by sequences complementary to the primers of either the P. pachyrhizi or P. meibomiae primary assay but hybridizing to a unique fluorogenic probe; the third contains unique primer- and probe-binding sequences, and was prepared as a cloned DNA fragment in a linearized plasmid. These ICs neither qualitatively nor quantitatively affected their primary assays. PpaIC and PmeIC were shown to successfully identify false-negative reactions resulting from endogenous or exogenous inhibitors, and can be readily adapted to function in a variety of diagnostic Q-PCR assays; the plasmid was found to successfully validate true negatives in similar Q-PCR assays for other soybean pathogens, as well as to function as a tracer molecule during DNA extraction and recovery.

Pitfalls in patch testing

Allergic contact dermatitis is a common skin disease affecting both genders and individuals of any age. The patch test is the only useful in vivo method for the etiological diagnosis of allergic contact dermatitis. It is an apparently simple technique, but pitfalls may be encountered. False-positive and false-negative reactions are the most common. Times of occlusion and of reading may influence the result. Some difficulties are encountered in patients with active dermatitis or under immunosuppressive therapy, and climate conditions may interfere with the procedure. In addition, difficulties may be encountered in performing the patch test on dark-skinned patients, and patch testing in children may give some misconception due to the fact that the technique has been optimized in adults. A validated standard for readings and the interpretation of the patch test result is a recommended goal.

Sensitivity and specificity of the nickel spot (dimethylglyoxime) test

The accuracy of the dimethylglyoxime (DMG) nickel spot test has been questioned because of false negative and positive test reactions. The EN 1811, a European standard reference method developed by the European Committee for Standardization (CEN), is fine-tuned to estimate nickel release around the limit value of the EU Nickel Directive from products intended to come into direct and prolonged skin contact. Because assessments according to EN 1811 are expensive to perform, time consuming, and may destruct the test item, it should be of great value to know the accuracy of the DMG screening test. To evaluate the sensitivity and specificity of the DMG test. DMG spot testing, chemical analysis according to the EN 1811 reference method, and X-ray fluorescence spectroscopy (XRF) were performed concomitantly on 96 metallic components from earrings recently purchased in San Francisco. The sensitivity of the DMG test was 59.3% and the specificity was 97.5% based on DMG-test results and nickel release concentrations determined by the EN 1811 reference method. The DMG test has a high specificity but a modest sensitivity. It may serve well for screening purposes. Past exposure studies may have underestimated nickel release from consumer items.

Evaluation of single and double-locus real-time PCR assays for methicillin-resistant Staphylococcus aureus (MRSA) surveillance

BackgroundMethicillin-resistant Staphylococcus aureus (MRSA) is a human pathogen, representing an infection control challenge. Conventional MRSA screening takes up to three days, therefore development of rapid detection is essential. Real time-PCR (rt-PCR) is the fastest method fulfilling this task. All currently published or commercially available rt-PCR MRSA assays relay on single or double-locus detection. Double-locus assays are based on simultaneous detection of mecA gene and a S. aureus-specific gene. Such assays cannot be applied on clinical samples, which often contain both coagulase-negative staphylococci (CoNS) and S. aureus, either of which can carry mecA. Single-locus assays are based on detection of the staphylococcal cassette chromosome mec (SCCmec) element and the S. aureus-specific orfX gene, assuming that it is equivalent to mecA detection.FindingsParallel evaluation of several published single and double-locus rt-PCR MRSA assays of 150 pure culture strains, followed by analysis of 460 swab-derived clinical samples which included standard identification, susceptibility testing, followed by PCR detection of staphylococcal suspected isolates and in-PCR mixed bacterial populations analysis indicated the following findings.Pure cultures analysis indicated that one of the single-locus assay had very high prevalence of false positives (Positive predictive value = 77.8%) and was excluded from further analysis. Analysis of 460 swab-derived samples indicated that the second single-locus assay misidentified 16 out of 219 MRSA's and 13 out of 90 methicillin-sensitive S. aureus's (MSSA) were misidentified as MRSA's. The double-locus detection assay misidentified 55 out of 90 MSSA's. 46 MSSA containing samples were misidentified as MRSA and 9 as other than S. aureus ending with low positive predicted value (<85%) and very low specificity (<62%).ConclusionThe results indicate that high prevalence of false-positive and false-negative reactions occurs in such assays.

A NOVEL MULTIPLEX POLYMERASE CHAIN REACTION FOR SIMULTANEOUS DETECTION OF YERSINIA ENTEROCOLITICA, STAPHYLOCOCCUS AUREUS, AEROMONAS AND SALMONELLA FROM CHICKEN MEAT AND MILK SAMPLES

ABSTRACTYersinia enterocolitica, Staphylococcus aureus, Aeromonas and Salmonella are among the most important foodborne bacterial pathogens. The majority of human infections caused by all of these organisms are associated with ingestion of undercooked and contaminated meat, dairy products and water where in the secreted bacterial toxins lead to foodborne intoxications. We, here, report a new multiplex polymerase chain reaction (mPCR) assay for the simultaneous detection of these important foodborne bacterial pathogens. The mPCR targeted Ail and virF genes of Y. enterocolitica, nuc and entB genes of S. aureus, aerA and 16S rRNA genes of Aeromonas and invA, an invasion protein A gene of Salmonella. An internal amplification control designed to check the false negative reactions in mPCR was also included. This procedure could detect initial populations of 1–100 cfu/g or /mL within 24 h in experimentally spiked food and water samples. When evaluated on a total of 104 naturally occurring food samples, the mPCR detected two samples to contain S. aureus, one was identified to contain Y. enterocolitica and four samples were identified to contain Salmonella species individually. This was compared with the standard microbiological and biochemical identification procedures.PRACTICAL APPLICATIONSAll the microorganisms selected in this study are food and waterborne and contaminate a variety of food items. Pathogenic Y. enterocolitica and Aeromonas species are able to grow and multiply and secrete toxins even at low temperatures. The high throughput and cost‐effective multiplex polymerase chain reaction method reported here could be a viable alternative for detection of pathogenic Y. enterocolitica, S. aureus, Aeromonas and Salmonella from food and environmental samples.

Understanding the cause of false negative β-d-glucuronidase reactions in culture media containing fermentable carbohydrate

To explain the basis for false negative beta-glucuronidase reactions seen with culture media containing lactose as a carbon and energy source. Escherichia coli strains were assessed for their reactions in culture media containing a beta-d-glucuronidase substrate either with or without lactose. An assay was developed to test for the expression of beta-D-glucuronidase at pH 5.0 and pH 7.2. Strains of E. coli that gave false negative glucuronidase reactions on media containing lactose generally expressed lower concentrations of the enzyme beta-D-glucuronidase than strains that gave positive results, although the difference was by no means consistent. Most strains that were negative on lactose-containing media expressed virtually no beta-D-glucuronidase activity at pH 5.0. Examination of colonies on Membrane lactose glucuronide agar (MLGA) from lightly polluted water showed that c. 10% of the E. coli present failed to yield green colonies on MLGA. E. coli that failed to produce green colonies on MLGA produced lower levels of beta-D-glucuronidase than did strains that formed green colonies, the difference being greater at pH 5.0 than pH 7.2. The false negative rate for E. coli 10% which is similar to that experienced in the study that originally described MLGA. Strains of E. coli that fail to produce typical colonies on MLGA might produce lower concentrations of the enzyme beta-D-glucuronidase. Whilst the enzyme activity is sufficient to be detected at pH 7.2, fermentation of lactose significantly lowers the pH of the medium and can result in reduced enzyme activity and therefore lack of detection. The false negative rate of c. 10% would be difficult to detect in routine laboratories as it would represent 1% or less of yellow colonies being identified as E. coli (assuming E. coli accounts for 10% of the total coliform population in drinking water).

A versatile internal control for use as DNA in real-time PCR and as RNA in real-time reverse transcription PCR assays

To develop and evaluate a TaqMan-based internal amplification control (IAC) that can be used as DNA in real-time PCR (qPCR) or as RNA in reverse transcription real-time PCR (qRT-PCR) to identify the presence of assay inhibition and to evaluate its incorporation into existing qPCR and qRT-PCR methods for bacterial detection. A DNA IAC was constructed by generating a 198-bp random sequence that was synthesized and inserted into a pZErO-2 vector and transformed into Escherichia coli. The RNA IAC was generated through in vitro transcription of the DNA IAC. Both IAC formats were tested individually in singleplex TaqMan reactions and also included in existing multiplex assays. The DNA IAC was incorporated in a Shigella spp. detection qPCR assay (targeting ipaH). The RNA IAC was successfully evaluated in a Salmonella spp. detection qRT-PCR (using invA mRNA as target). A highly versatile IAC that can be supplemented to qPCR and qRT-PCR pathogen detection methods was developed, greatly reducing the confounding effects of false negatives because of PCR inhibitors without affecting pathogen detection. The frequency of false negatives associated with qPCR analyses is prevalent in certain matrices, particularly those involving complex foods. Hence, the IAC presented here provides a solution to unforeseen false-negative reactions in PCR.

Validation of a hospital‐laboratory workstation for immunohematologic methods

The FREELYS Nano system (Diagast) is a manual workstation for ABO/D grouping, Rh phenotyping, K typing, and antibody screening (ABS) for immunoglobulin G (IgG) antibodies only and works with the erythrocyte-magnetized technology (EMT). The principle of EMT is based on magnetization of red blood cells and avoids centrifugation and washing steps. A total of 304 samples were tested with our routine blood bank methods, 100 samples for ABO/D grouping, 196 samples (100 at first evaluation, 96 at second evaluation) for Rh phenotyping and K typing (PK7200, Olympus), and 108 samples for ABS (DiaMed). All samples were tested in parallel with the FREELYS Nano. We found a 100% concordance between the observed (FREELYS Nano) and the expected (Olympus PK7200) results for ABO/D grouping in all 100 samples. For Rh phenotyping and K tests, in 24 of 100 samples false-positive reactions were observed in the first evaluation by the FREELYS Nano. After changing the test kit batch for Rh phenotyping by the manufacturer, a complete concordance in Rh phenotyping and K tests was observed in a second evaluation. For ABS, the FREELYS Nano showed in 4 of 108 samples (3.7%) false-negative reactions for IgG antibodies (two anti-K, one anti-E, one anti-C(w)), and one (0.9%) false-positive reaction. The FREELYS Nano is reliably suited to ABO/D grouping, Rh phenotyping, and K testing. The rate of false-negative reactions for IgG antibodies should be reduced.

Humoral immunity in tuberculin skin test anergy and its role in high-risk persons exposed to active tuberculosis

The most common test to identify latent tuberculosis is the tuberculin skin test that detects T cell responses of delayed type hypersensitivity type IV. Since it produces false negative reactions in active tuberculosis or in high-risk persons exposed to tuberculosis patients as shown in this report, we studied antibody profiles to explain the anergy of such responses in high-risk individuals without active infection.Our results showed that humoral immunity against tuberculin, regardless of the result of the tuberculin skin test is important for protection from active tuberculosis and that the presence of high antibody titers is a more reliable indicator of infection latency suggesting that latency can be based on the levels of antibodies together with in vitro proliferation of peripheral blood mononuclear cells in the presence of the purified protein derivative. Importantly, anti-tuberculin IgG antibody levels mediate the anergy described herein, which could also prevent reactivation of disease in high-risk individuals with high antibody titers. Such anti-tuberculin IgG antibodies were also found associated with blocking and/or stimulation of in vitro cultures of PBMC with tuberculin. In this regard, future studies need to establish if immune responses to Mycobacterium tuberculosis can generate a broad spectrum of reactions either toward Th1 responses favoring stimulation by cytokines or by antibodies and those toward diminished responses by Th2 cytokines or blocking by antibodies; possibly involving mechanisms of antibody dependent protection from Mtb by different subclasses of IgG.